Mutational analysis and molecular modelling of the antagonist SR 144528 binding site on the human cannabinoid CB2 receptor

doi:10.1016/S0014-2999(00)00439-8

European Journal of Pharmacology

Volume 401, Issue 1, 28 July 2000, Pages 17-25

https://doi.org/10.1016/S0014-2999(00)00439-8 Get rights and content

Abstract

We have investigated the binding site of the subtype specific antagonist SR 144528, (N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methoxybenzyl)-pyrazole-3-carboxamide) on the human cannabinoid CB₂ receptor based on functional studies with mutated receptors. Two serine residues in the fourth transmembrane region, Ser¹⁶¹ and Ser¹⁶⁵, were singly mutated to the cognate cannabinoid CB₁ receptor residue, alanine, and each gave receptors with wild-type properties for the cannabinoid agonists CP 55,940 (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol) and WIN 55212-2 (R)-(+)[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl) methanone, which SR 144528 completely failed to antagonise. Molecular modelling studies show that SR 144528 interacts with residues in transmembrane domains 3, 4, and 5 of the cannabinoid CB₂ receptor through a combination of hydrogen bonds and aromatic and hydrophobic interactions. In addition, the replacement by serine of a nearby cannabinoid CB₂ receptor-specific residue, Cys¹⁷⁵ resulted in wild-type receptor properties with CP 55,940, loss of SR 144528 binding and eight-fold reduced binding and activity of WIN 55212-2, a result compatible with a recently-proposed binding site model for WIN 55212-2.

Introduction

The active constituent in Cannabis sativa, Δ⁹-tetrahydrocannabinol, the endogenous cannabinoids N-arachidonoylethanolamine (anandamide) (Devane et al., 1992) and 2-arachidonylglycerol (Mechoulam et al., 1995) together with numerous synthetic cannabinoid agonists (Pertwee, 1997) exert their effect through the mediation of two 7-transmembrane domain G protein-coupled receptors. The cannabinoid CB₁ receptor Gérard et al., 1991, Matsuda et al., 1990 is expressed principally in the brain, but is also found in peripheral tissues, whereas the cannabinoid CB₂ receptor (Munro et al., 1993), absent from the brain, is found predominantly in blood cells (Galiègue et al., 1995). The two subtypes share only 44% homology, which has facilitated the development of the highly-specific and potent antagonists, SR 141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride) (Rinaldi-Carmona et al., 1994) and SR 144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methoxybenzyl)-pyrazole-3-carboxamide) (Rinaldi-Carmona et al., 1998) for the cannabinoid CB₁ and cannabinoid CB₂ receptor, respectively (Fig. 1). SR 144528 has a 700-fold higher affinity for the cannabinoid CB₂ receptor than for the cannabinoid CB₁ receptor (Rinaldi-Carmona et al., 1998). Through competition binding experiments, we recently showed that residues important for the subtype specificity of SR 144528 for the cannabinoid CB₂ receptor were located somewhere within the transmembrane domain 4–extracellular loop 2–transmembrane domain 5 region (Shire et al., 1999), in the same region previously found to be important for binding of SR 141716A to the cannabinoid CB₁ receptor (Shire et al., 1996a). That report was mainly based on competition binding experiments with the tritiated bicyclic cannabinoid agonist CP 55,940, (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol) (Fig. 1) which has comparable binding affinities for the cannabinoid CB₁ and cannabinoid CB₂ receptors and, as we found, for hybrid constructs of the two receptors. Another powerful cannabinoid agonist, WIN 55212-2, (R)-(+)[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl) methanone (Fig. 1) has a ∼20-fold higher binding affinity for the human cannabinoid CB₂ receptor than for the human cannabinoid CB₁ receptor Shire et al., 1996b, Showalter et al., 1996. The binding site for WIN 55212-2 appeared to be in the same region of the human cannabinoid CB₂ receptor as that of the SR compounds (Shire et al., 1999) and this was recently confirmed by the discovery that Phe¹⁹⁷ in transmembrane region 5 was crucial for the subtype specificity of WIN 55212-2 (Song et al., 1999).

Here, we describe experiments with monkey kidney COS-7 cells expressing point mutated cannabinoid CB₂ receptors that led to the discovery of two residues in transmembrane domain 4, Ser¹⁶¹ and Ser¹⁶⁵ that are implicated in the binding of SR 144528, but not in that of either CP 55,940 or WIN 55212-2, confirmed by both binding and activity analysis. Mutation of a third residue, Cys¹⁷⁵ also eliminated SR 144528 binding, diminished WIN 55212-2 binding and activity, but had no effect on CP 55,940 binding affinity or activity. From the results obtained and using molecular modelling techniques, we propose a model for SR 144528 docked in the cannabinoid CB₂ receptor.

Section snippets

Drugs and chemicals

WIN 55212-2 was purchased from Sigma (Saint-Quentin-Fallavier, France). CP 55,940, SR 141716A, and SR 144528 were synthesised in the Chemistry Department of Sanofi (Montpellier, France). Drugs were dissolved in dimethyl sulfoxide, the final solvent concentration in assays never exceeding 0.1% (v/v), an amount without effect on ligand binding. Bifluor liquid scintillant and [³H]CP 55,940 (111.9 Ci/mmol) were from New England Nuclear (Paris, France). Lipofectamine was from Gibco/BRL Life

Binding characteristics of the wild-type and mutated CB₂ receptors and effect on luciferase stimulation by CP 55,940, WIN 55212-2, and SR 144528

We previously reported that the mutation of each of the conserved cannabinoid CB₂ receptor extracellular loop 2 cysteines Cys¹⁷⁴ and Cys¹⁷⁹ (Fig. 2) to serine resulted in receptors that failed to bind [³H]CP 55,940 (Shire et al., 1996a). However, immunofluorescence examination of intact cells by means of the amino terminal c-myc epitope confirmed that the receptors were present on the cell surface (Shire et al., 1996a). In the present study, we found that the C174S and C179S mutants also failed

Discussion

In the model for SR 144528 docked into the cannabinoid CB₂ receptor presented here, we propose that the specificity of the molecule relies on crucial contacts with subtype specific residues in transmembrane domain 4. Other contacts are with mainly conserved residues in transmembrane domains 3 and 5. This corroborates previous work in which we used chimeric cannabinoid CB₁/CB₂ receptor to show that an important contribution to the high affinity binding sites for SR 144528 and SR 141716A was

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Specifically, five hydrophobic residues were found to be in close contact with all three ligands during docking simulations: V113, L192, L201, V261 and W258. Indeed, L201, V261 and W258 have previously been implicated in the formation of the ligand binding pocket [23,39]. A prior report affirms that residue 3.32 is occupied by a negatively charged amino acid in biogenic amine receptors (D2 Dopamine, ß2 Adrenergic and m3 Muscarinic) and that it is essential for recognition and binding of endogenous amines [40].
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Mutational analysis and molecular modelling of the antagonist SR 144528 binding site on the human cannabinoid CB2 receptor

Abstract

Introduction

Section snippets

Drugs and chemicals

Binding characteristics of the wild-type and mutated CB2 receptors and effect on luciferase stimulation by CP 55,940, WIN 55212-2, and SR 144528

Discussion

J. Biol. Chem.

Eur. J. Pharmacol.

FEBS Lett.

J. Mol. Biol.

J. Biol. Chem.

Biochem. Pharmacol.

Pharmacol. Ther.

FEBS Lett.

J. Biol. Chem.

Biochim. Biophys. Acta Gene Struct. Expression

Life Sci.

J. Mol. Graphics

J. Biol. Chem.

A critical role for a tyrosine residue in the cannabinoid receptors for recognition of several compounds excluding the indoles

Isolation and structure of a brain constituent that binds to the cannabinoid receptor

Science

Mutational analysis and molecular modelling of the antagonist SR 144528 binding site on the human cannabinoid CB₂ receptor

Binding characteristics of the wild-type and mutated CB₂ receptors and effect on luciferase stimulation by CP 55,940, WIN 55212-2, and SR 144528